Considerable progress has been made in elucidating the critical regulatory role of cullin-based E3 ubiquitin ligases over a wide array of biological processes in eukaryotic cells. However, we are far from understanding their intricacies and we have only begun to appreciate their complex regulatory networks. While defective regulation of the cullin-dependent proteolysis pathways is manifest in human diseases including cancers, we are still in our infancy with respect to developing effective pharmacologic agents due to insufficient mechanistic understanding of these processes. Thus, if we are to understand aberrant growth, we must have fundamental knowledge of how cullins mediate proteolysis and how these processes are regulated. Recently, we have identified a novel member of the cullin protein family called CUL7, containing both the cullin and DOC signature domains. Remarkably, CUL7 assembles an SCF-like E3 Ub ligase complex composed of Skp1, CUL7, ROC1, and a previously uncharacterized F-box protein, Fbx29. We propose to elucidate the biological function of the CUL7/Fbx29-based E3-1ike complex in mediating protein degradation by identifying substrate(s) of Fbx29. Nedd8 is a specific cullin modifier that up-regulates the activity of cullin-based E3 Ub ligases. Recently, we have cloned a potential regulator of the Nedd8 pathway called hDEN-1, a novel Nedd8 isopeptidase. We will investigate the biological function of hDEN-1 in Ub-dependent protein degradation pathways by assessing the function of hDEN-1 in regulating Nedd8-dependent SCF activities in mammalian cells, as well as by examining the biological role of this novel protease using S. pombe and Drosophila model systems. The pVHL tumor suppressor functions by assembling a cullin 2-containing E3 ligase that targets HIF-1alpha for ubiquitination and degradation. Recent studies have revealed that a novel post-translational modification, prolyl hydroxylation, plays a central role in regulating the pVHL/HIF-1 pathway. Hydroxylation of HIF-1alpha at proline residue 564, by the PHO1/SM20 family prolyl hydroxylase, is required for its interaction with pVHL to initiate the ubiquitination. More recently, we have identified a cellular activity that stimulates the function of the PHD1/SM20 prolyl hydroxylase. We propose to determine the molecular identity of this novel activity, which may prove to be yet another critical regulator of the pVHL/HIF-1 pathway.